Pegylated hemoglobin and albumin and uses thereof

ABSTRACT

The present invention provides PEGylated hemoglobins and PEGylated albumins comprising polyethylene glycol (PEG) conjugated to hemoglobin or to albumin, wherein the PEG is a maleimide PEG, an alkylamide PEG, an iodoacetamide PEG, a p-nitro thio-phenyl PEG, a vinyl sulfone PEG, or a mixed disulfide PEG; and PEGylated albumins and PEGylated hemoglobins comprising polyethylene glycol (PEG) attached to a thiolated amino group of albumin or hemoglobin, wherein the amino group is thiolated using dithio sulfo succinimidyl propionate (DTSSP) or dithiosuccinimidyl propionate (DTSP) or dithiobispropionimidate. The invention also provides methods of preparing PEGylated hemoglobins and PEGylated albumins comprising a) reacting hemoglobin or albumin with a thiolating agent and with a PEGylating agent, and b) capping unPEGylated reactive thiols of hemoglobin or albumin with N-ethyl maleimide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 60/689,175, filed Jun. 10, 2005, the content of which isherein incorporated by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with United States government support underNational Institutes of Health (NIH) grant numbers HL58247 and HL71064,and U.S. Army grant PR023085. Accordingly, the United States governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to inparenthesis. Full citations for these references may be found at the endof the specification immediately preceding the claims. The disclosuresof these publications are hereby incorporated by reference in theirentireties into the subject application to more fully describe the artto which the subject application pertains.

Conjugation of polymers to peptide and protein therapeutics to generatehybrid molecules with unique and distinct molecular properties hasbecome a popular approach to alter and/or control their stability,biodistribution, pharmacokinetics and toxicology. Since the pioneeringwork of Abuchowski et al. (1997a & b) of grafting polyethylene glycol(PEG) chains to albumin, PEGylation has been a very widely usedconjugation approach to generate protein-polymer bio-conjugates ofunique biological properties or diminished toxicities.

The first step of the PEGylation of proteins and peptides can involvefunctionalizing PEG with group specific reagents, so that theconjugation of PEG to protein can be targeted to specific side chaingroups of the proteins, such as amino, carboxyl, sulfhydryl or guanidinogroups. More recently, the strengths of site directed mutagenesis havealso been integrated to achieve site specific PEGylation. Cysteine (Cys)residues can be introduced in a site specific fashion in place ofpreselected surface amino acid residues of proteins. The thiol groups ofthe newly introduced Cys residues can be targeted for PEGylation usingmaleimide chemistry based PEG reagents. Replacement of Serine (Ser) orThreonine (Thr) with Cys has an advantage that the net charge of themutant protein is not altered as a result of the PEGylation, i.e. aconservative PEGylation protocol as far as the site directed mutagenesisof the parent protein is conservative.

A chemical approach to introduce new thiols on the ε-amino groups ofproteins as a means of increasing accessibility of the surface aminogroups for PEGylation and targeting the PEG reagents to these sites bymaleimide chemistry has been developed (Acharya et al., 1996). Theinitial approach involved thiolation of amino groups of proteins using2-iminothiolane. In a preferred protocol, protein is incubated withiminothiolane in the presence of PEG maleimide, and the new thiol groupsgenerated in situ on the protein amino groups are trapped immediately byPEG maleimide as succinimidyl derivatives (Acharya et al., 2003).

Hemoglobin (Hb) based blood substitutes are being developed to overcomeshortages of blood supply (Chang, 1999; Klein, 2000). The mostextensively studied and financed blood substitute has been diaspirincross-linked Hb. Though intramolecular crosslinking of Hb helped toovercome nephrotoxicity and high oxygen affinity of acellular Hb, thetwo major limitations of stroma-free Hb as a blood substitute (Chang,1999), the product remained vasoactive (Alayash et al., 2001; Kramer,2003; Winslow, 2000). The vasoactivity has been attributed to theextravasation of acellular Hb and the scavenging of nitric oxide (NO) bythe extravasated Hb.

Enhancing the molecular size of Hb by oligomerization to prevent orreduce the extravasation of Hb has been one of the solutions advanced toovercome the vasoactivity of acellular Hb, while another is to lower theaffinity of Hb to nitric oxide by site directed mutagenesis. Animalstudies have shown that both approaches reduce the pressor effect of Hb(Gulati et al., 1999).

An alternate approach to overcome the vasoactivity of Hb involvesengineering the properties of plasma volume expanders into Hb, namelyhigh viscosity and high colloidal osmotic pressure to Hb (Acharya etal., 2005). Enzon PEGylated bovine Hb, which carries ten copies ofPEG-5K chains, was found to be nonhypertensive even though its affinityfor NO is comparable to that of other modified Hbs that are underclinical trial. The unusual molecular properties of Enzon PEGylatedbovine Hb, namely enhanced molecular volume, high viscosity and highcolloidal osmotic pressure, which are also the properties of plasmavolume expanders, have been attributed as the molecular basis of theneutralization of vasoactivity of acellular Hb. Accordingly, PEGylationof Hb has been considered as an approach to generate non-hypertensiveHb. This application of polyethylene glycol (PEG), a water-soluble,inert and nontoxic polymer, reflects a very different translation ofPEGylation than other applications of other molecular properties inducedto proteins by PEGylation, where PEGylation-induced increasedsolubility, increased half-life, and reduced access of molecular surfaceto the immune system are used to generate PEG-protein conjugates oftherapeutic value (Bailon and Berthold, 1998; Harris et al., 1997,2003).

In an attempt to establish that the neutralization of vasoactivity is ageneralized consequence of PEGylation induced molecular properties ofacellular Hb and not unique to Enzon PEGylated bovine Hb, ahexaPEGylated human Hb [(SP-PEG-5K)₆-Hb] was generated using aPEGylation platform referred to as extension arm facilitated PEGylationprotocol (Acharya et al., 2005; Manjula et al., 2005). ThishexaPEGylated Hb exhibited an unusually high increase in the molecularvolume, increased viscosity and higher colloidal osmotic pressure andwas non-hypertensive.

The non-hypertensive PEGylated human Hb, (SP-PEG5K)₆-Hb, carries onlysix copies of PEG-5K chains while the Enzon PEGylated bovine Hb carriesten copies of PEG-5K chains. In addition, the chemistry of conjugationof PEG-chains in the two products is very distinct. In the decaPEGylatedbovine Hb of Enzon, the PEG-chains are conjugated to the surface aminogroups of Hb through an urethane linkage, which results in the loss ofthe positive charge of the E-amino group of Lysine (Lys) residues towhich PEG chains are conjugated. In the hexaPEGylated human Hb, thePEG-5K chains are conjugated to the surface amino groups using theextension arm facilitated PEGylation. In this protocol, the surfaceamino groups are first reacted with iminothiolane, which results in theextension of the side chain of Lys residues by the linking of δ-mercaptobutirimidyl chains, and the thiol groups of the extension arm aremodified with maleimide PEG (Acharya et al., 2005; Manjula et al. 2005).The higher efficiency of the PEG-chains of hexaPEGylated Hb toneutralize the vasoactivity could be a correlate of the fact that theextension arm facilitated PEGylation conjugates the PEG-chains withoutchanging the surface charge of Hb, i.e. the PEGylation is conservative.Since the colloidal osmotic pressure (COP) of the hexaPEGylated Hb iscomparable to that of decaPEGylated Hb, the results reflect the role ofthe conservation of the positive charge of Hb at the sites ofPEGylation. When the surface charge is conserved, the PEG-chainsconjugated are possibly more efficient in inducing the desirablemolecular properties to Hb that facilitates the neutralization of itsvasoactivity.

To gain further insight into the possible advantages of conserving thesurface charges at the site of the conjugation, another hexaPEGylated Hbhas been generated in which the charge of the surface amino groups isconserved. {acute over (ω)}-methoxy PEG 5K-propoinaldehyde is conjugatedto Hb in the presence of sodium cyanoborohydride (reductive alkylationchemistry). The molecular properties of this PEG-Hb conjugate,particularly the COP of (Propyl-PEG5K)₆-Hb was considerably higher thanthat of [(SP-PEG-5K)₆-Hb], leading to the suggestion that either thechemistry of conjugation of PEG-chains to Hb or the site selectivity ofPEGylation of Hb influences the molecular properties of the PEG-Hb (Huet al., 2005).

SUMMARY OF THE INVENTION

The present invention provides PEGylated hemoglobins and PEGylatedalbumins comprising a polyethylene glycol (PEG) conjugated to hemoglobinor to albumin, wherein the PEG is a maleimide PEG, an alkylamide PEG, aniodoacetamide PEG, a p-nitro thio-phenyl PEG, a vinyl sulfone PEG, or amixed disulfide PEG.

The invention also provides a PEGylated albumin or a PEGylatedhemoglobin comprising a polyethylene glycol (PEG) attached to athiolated amino group of albumin or hemoglobin, wherein the amino groupis thiolated using dithio sulfo succinimidyl propionate (DTSSP) ordithiosuccinimidyl propionate (DTSP) or dithiobispropionimidate.

The invention provides a method of preparing a PEGylated hemoglobin or aPEGylated albumin comprising: a) reacting hemoglobin or albumin with athiolating agent to produce thiolated hemoglobin or thiolated albumin;b) reacting the thiolated hemoglobin or the thiolated albumin with aPEGylating agent; and c) capping unPEGylated reactive thiols ofhemoglobin or albumin with a capping agent.

The invention further provides a method of preparing a PEGylatedhemoglobin or a PEGylated albumin comprising: a) reacting hemoglobin oralbumin with dithio sulfo succinimidyl propionate (DTSSP) or withdithiosuccinimidyl propionate (DTSP) or with dithiobispropionimidate tothiolate the hemoglobin or albumin, and b) reacting the thiolatedhemoglobin or the thiolated albumin with a PEGylating agent.

The invention still further provides compositions and blood substitutes(plasma volume expanders) comprising PEGylated hemoglobins and PEGylatedalbumins and methods of treating a subject which comprise administeringto the subject any of the PEGylated hemoglobins or PEGylated albuminsdisclosed herein.

Additional objects of the invention will be apparent from thedescription which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of thiolation mediated conservativePEGylation of proteins. The thiol groups of the thiolated proteingenerated as an intermediate in this protocol can be the target sitesfor PEG reagents with sulfhydryl group specific functional groups.PEGylation of the protein based on the reaction of monofunctional PEGmaleimide and monofunctional iodoacetamido PEG have been used asspecific examples to demonstrate the conservative PEGylation reaction.

FIG. 2A-2B. Characterization of (Propyl-PEG5K)₆-Hb and(Propyl-PEG5K)₆-αα-Hb by SDS-PAGE (A) and isoelectric focusing (IEF)(B). A: SDS-PAGE was carried out on a precast 14% tris-glycine gel fromthe Invitrogen Corporation. Lane 1, molecular weight markers; Lane 2,HbA; Lane 3, αα-Hb; Lane 4, (Propyl-PEG5K)₆-αα-Hb; and Lane 5,(Propyl-PEG5K)₆-Hb. B: IEF was operated using precast resolve gels fromIsolab and a blend of pH 6-8 resolve ampholytes. Lane 1, HbA; Lane 2,αα-Hb; Lane 3, (Propyl-PEG5K)₆-Hb; and Lane 4, (Propyl-PEG5K)₆-αα-Hb.

FIG. 3. Size exclusion chromatographic analysis of PEGylated Hb samples.The analysis was carried out at room temperature on two HR10/30 Superose12 columns (Amersham-Pharmacia Biotech) connected in series. The columnwas eluted with PBS, pH 7.4 at a flow rate of 0.5 ml/min, and theeffluent was monitored at 540 nm.

FIG. 4A-4B. Influence of PEG-chain length on the molecular volume of(Propyl-PEG)₆ αα-Hb. A: Size exclusion chromatographic analysis ofPEGylated protein. The analysis was carried out at room temperature ontwo HR10/30 Superose 12 columns connected in series. The column waseluted with PBS, pH 7.4 at a flow rate of 0.5 ml/min. B: Sizeenhancement of Hb as a function of the length of attached PEG chains.Molecular radii were measured by dynamic light scattering at a proteinconcentration of 1 mg/ml. ΔV was calculated with an equation ΔV=4π(R³−R₀³)/3. R and R₀ are radii of PEGylated Hbs and HbA, respectively.

FIG. 5. Colloidal osmotic pressures of HbA (▪), (Propyl-PEG5K)₆-Hb (),(Propyl-PEG5K)₆-αα-Hb (▴) as a function of protein concentration. Aseries of concentrations of HbA samples were measured by a Wescor 4420Colloidal Osmometer in PBS (pH 7.4) and at room temperature. The insetindicated the comparison of the viscosity of (Propyl-PEG5K)₆-αα-Hb withthat of (Propyl-PEG5K)₆-Hb at 4 g/dL.

FIG. 6. S_(20,W) of PEGylated proteins as a function of hemoglobinconcentrations. Sedimentation velocity measurements of(Propyl-PEG5K)₆-Hb (□), (Propyl-PEG5K)₆-αα-Hb (◯), HbA (▪) and αα-Hb (•)were conducted in a Beckman XL-I analytical ultracentrifuge in PBSbuffer at pH 7.4, 25° C. and 55,000 rpm. Boundary movement was followedat 405 nm using the centrifuge's absorption optics.

FIG. 7A-7B. Circular dichroism spectra of PEGylated proteins. Circulardichroism spectra of Hb samples were recorded at 25° C. with a 0.2-cmlight path cuvette (310 μl) in far-UV region (200-250 nm, A), near-UVand Soret region (250-480 nm, B), and visible region (480-650 nm). Themolar ellipticity, θ, is expressed in deg·cm²/dmol on a heme basis.

FIG. 8. Intrinsic fluorescence emission spectra of HbA, αα-Hb,(Propyl-PEG5K)₆-Hb and (Propyl-PEG5K)₆-αα-Hb. The excitation wavelengthwas 280 nm. The measurements were performed using Shimadzuspectrofluorimeter at room temperature. All the samples used were at Hbconcentration of 1 mg/ml in PBS, pH 7.4.

FIG. 9. Comparison of the SEC chromatography of the hexaPEGylated Hbgenerated from uncrosslinked and alpha alpha fumaryl Hb.

FIG. 10. Kinetics of thiolation of (Thiocarbamoyl Phenyl PEG-5K) humanserum albumin.

FIG. 11. Schematic representation of thiolation mediatednon-conservative PEGylation of Proteins. The thiol groups are generatedon the protein by reaction with bis succinimidyl dithiopropionimidtaefollowed by reduction of the disulfide bonds with tris carboxyethylphosphine and then subjected to PEGylation either using maleimide PEG oriodoacetamido PEG just as in the thiolation mediated conservativePEGylation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a PEGylated hemoglobin or aPEGylated albumin comprising a polyethylene glycol (PEG) conjugated tohemoglobin or to albumin, wherein the PEG is a maleimide PEG, analkylamide PEG, an iodoacetamide PEG, a p-nitro thio-phenyl PEG, a vinylsulfone PEG, or a mixed disulfide PEG.

As used herein, “PEGylation” means linking to polyethylene glycol (PEG),and a “PEGylated” hemoglobin or albumin is a hemoglobin or albumin thathas PEG conjugated to it.

In a preferred embodiment, the PEGylated hemoglobin is acarboxamidomethyl (CAM) PEGylated hemoglobin.

The maleimide PEG can be, for example, a maleimide PEG comprising analkyl linker or, preferably, a maleimide phenyl PEG. The PEG can beattached to albumin or hemoglobin via a linker and/or an extension arm.As used herein, an “extension arm” refers to the carbon chain-thiolgroup that is attached to albumin or hemoglobin during a thiolationprocess. The extension arm places the thiol group away from the surfaceof the albumin or hemoglobin, thereby enhancing the accessibility of thethiol group to bulky PEG reagents. The linker may comprise an alkyl,aryl and/or heteroaryl group. For example, the alkyl group can be apropyl group, and the aryl group can be a phenyl group. The linker orextension arm may comprise a δ-mercapto butyrimidyl chain or aγ-mercapto propylamide chain.

The invention also provides a PEGylated albumin or a PEGylatedhemoglobin comprising a polyethylene glycol (PEG) attached to athiolated amino group of albumin or hemoglobin, wherein the amino groupis thiolated using dithio sulfo succinimidyl propionate (DTSSP) ordithiosuccinimidyl propionate (DTSP) or dithiobispropionimidate.

The invention further provides thiocarbamoyl PEGylated albumin,PEGylated thiolated albumin, PEGylated polynitroxylated albumin, andmethods of preparing these PEGylated albumins.

Each PEG chain may have a molecular weight of 200 daltons to 20,000daltons, preferably 3,000 to 5,000 daltons, and more preferably 5,000daltons. PEGs of various molecular weights, conjugated to variousgroups, can be obtained commercially, for example from NektarTherapeutics, Huntsville, Ala.

In one embodiment, the PEGylated albumin has a molecular weight of about130 kDa. In one embodiment, the PEGylated albumin has a molecular radiusof 8-9 nm. There may be between 6-18 PEG chains conjugated to albumin.In one preferred embodiment, 12 PEG chains are conjugated to albumin.Preferably, the PEGylated albumin has a colloid osmotic pressure of37-40 mm Hg. Preferably, the PEGylated albumin has a viscosity of 2.0 to4.0 cP.

In one embodiment, the PEGylated hemoglobin has a molecular radius of5-6 nm. There may be between 2-8 PEG chains conjugated to hemoglobin. Ina preferred embodiment, 4-6 PEG chains are conjugated to hemoglobin.Preferably, the PEGylated hemoglobin has a colloid osmotic pressure of34-36 mm Hg. The PEGylated hemoglobin can have a viscosity of 2.0 to 4.0cP.

Preferably, the hemoglobin that is PEGylated contains an intramolecularcrosslink. Examples of intramolecular crosslinks include αα-crosslinkingand ββ-crosslinking. Preferably, crosslinking the hemoglobin increasesthe molecular volume of the PEGylated crosslinked hemoglobin.Preferably, crosslinking the hemoglobin decreases the colloidal osmoticpressure of the PEGylated crosslinked hemoglobin.

The PEGylation procedure can be selected so that PEGylation does notalter the surface charge of albumin or hemoglobin or so that PEGylationdoes alter the surface charge of albumin or hemoglobin.

Preferably, PEGylation does not impair drug binding ability of albumin.

The invention provides a method of preparing a PEGylated hemoglobin or aPEGylated albumin comprising:

a) reacting hemoglobin or albumin with a thiolating agent to producethiolated hemoglobin or thiolated albumin;

b) reacting the thiolated hemoglobin or the thiolated albumin with aPEGylating agent; and

c) capping unPEGylated reactive thiols of hemoglobin or albumin with acapping agent, such as, for example, N-ethyl maleimide.

The thiolating agent can be, for example, iminothiolane, dithio sulfosuccinimidyl propionate (DTSSP) or dithiosuccinimidyl propionate (DTSP)or dithiobispropionimidate. The PEGylating agent can be, for example, aniodoacetamide PEG or a maleimide PEG.

The invention further provides a method of preparing a PEGylatedhemoglobin or a PEGylated albumin comprising:

a) reacting hemoglobin or albumin with dithio sulfo succinimidylpropionate (DTSSP) or with dithiosuccinimidyl propionate (DTSP) or withdithiobispropionimidate to thiolate the hemoglobin or albumin, and

b) reacting the thiolated hemoglobin or the thiolated albumin with aPEGylating agent.

The PEGylating agent can be, for example, an iodoacetamide PEG or amaleimide PEG.

The methods can further comprise preparing a hemoglobin having anintramolecular crosslink. Examples of intramolecular crosslinkinginclude αα-crosslinking and ββ-crosslinking.

The invention still further provides a PEGylated hemoglobin or aPEGylated albumin prepared by any of the methods disclosed herein.

The invention provides for the use of any mammalian albumin orhemoglobin, such as, for example, human hemoglobin, human serum albumin,and bovine serum albumin.

The invention also provides a composition comprising any of thePEGylated hemoglobins or PEGylated albumins disclosed herein or preparedby any of the methods disclosed herein, and a pharmaceuticallyacceptable carrier. The invention further provides a blood substitute(plasma volume expander) comprising any of the PEGylated hemoglobins oralbumins disclosed herein or prepared by any of the methods disclosedherein. Pharmaceutically acceptable carriers include, but are notlimited to, saline, phosphate buffered saline, Ringer's solution,lactated Ringer's solution, Locke-Ringer's solution, Kreb's Ringer'ssolution, Hartmann's balanced saline solution, and/or heparinized sodiumcitrate acid dextrose solution. The pharmaceutical compositions of thepresent invention may be administered by conventional means includingbut not limited to transfusion and injection. The invention providesmethods of treating a subject which comprises administering to thesubject any of the PEGylated hemoglobins or PEGylated albumins disclosedherein.

The present invention is illustrated in the following ExperimentalDetails section, which is set forth to aid in the understanding of theinvention, and should not be construed to limit in any way the scope ofthe invention as defined in the claims that follow thereafter.

EXPERIMENTAL DETAILS I. Introduction

The PEGylation protocol, thiolation mediated maleimide chemistry(succinimidylation) based PEGylation, is schematically represented inFIG. 1. Reaction of the surface amino groups of the protein with2-iminothiolane results in the δ-mercapto butyrimidination of thereactive surface ε-amino groups of its Lys residues, thereby introducinga four carbon atom extension arm between the ε-amino groups and thenewly introduced thiol groups. The PEG maleimide then reacts with thethiol groups to conjugate the PEG-chains to the protein. Since thethiolating reagent, 2-iminothiolane, does not carry free thiol group andthiol groups are generated in situ on reaction of iminothiolane with theamino groups, the thiolating reagent and the PEG maleimide can beincubated together with the protein without any concern for theconsumption of the maleimide PEG by the thiolating reagent. Thisapproach is referred to as one step thiolation mediated maleimidechemistry based PEGylation of proteins. This approach is a preferred oneover the two step thiolation mediated maleimide chemistry basedPEGylation protocol, wherein thiolation is done in the absence ofmaleimide PEG and the PEGylation is carried out after the thiolation.Introduction of an ‘extension arm’ is a feature of this thiolationmediated PEGylation protocol. The strategy of introducing an extensionarm on the ε-amino groups of Lys residues of protein provides anincreased accessibility to the new thiol groups (as compared to theoriginal amino groups) of the macromolecular PEG-reagent.

II. Maleimide PEG and Iodoacetamide PEG based PEGylation of Hemoglobin

A modified protocol for PEGylation of Hb has been developed thatgenerates a product with four to six PEG-5K chains (average number) pertetramer. One step protocol for PEGylation of proteins has been thepreferred choice to overcome (or at least minimize) the increasedpotential of the thiolated Hb to participate in intermolecular crosslinking of the protein at the higher protein concentration that is usedin the new reaction conditions. In the new protocol, the PEGylation ofHb is carried out at a Hb concentration of 1 mM in the presence of 10 mMiminothiolane and 10 mM Maleimide phenyl PEG for 6 hours at 4° C. togenerate Hb-PEG conjugate. This Hb-PEG conjugate exhibits a molecularsize enhancement that is slightly higher than that of tetraPEGylated dogHb, but smaller than that of the hexaPEGylated Hb [(SP-PEG-5K)₆-Hb]generated by PEGylation of Hb at a concentration of 0.5 mM in thepresence of 5 mM iminothiolane and 10 mM maleimide phenyl PEG. Themolecular radius of the new product and the results of the globin chainanalysis clearly reflect that the new material carries less number ofPEG-5K chains per tetramer than the previously characterized product,[(SP-PEG5K)₆-Hb].

In the design and generation of the non-hypertensive, hexaPEGylated Hb,it has been assumed that all the thiol groups generated in situ on Hb onits reaction with iminothiolane are completely trapped by maleimide PEGas succinimidyl phenyl PEG-derivatives. But given the multiplicity ofthe sites of thiolation exposed by the tryptic peptide mapping of thePEGylated protein, it is conceivable that the Hb-PEG conjugate may carryunderivatized thiol groups. Recent studies indicated that some amount ofhigher molecular weight products are generated after storage at −80° C.over months. Since molecular size homogeneity is not a direct correlateof the site selectivity of thiolation, and appears to be a consequenceof a degree of resistance of PEGylated Hb with six copies of PEG-5Kchains to undergo further PEGylation, it is possible that at the end ofthe PEGylation reaction, the PEGylated Hb may still carry someunmodified thiols and these may be responsible for the molecular sizeheterogeneity generated during storage.

Accordingly, in developing this new protocol for generating Hb-PEG-5Kconjugate that carries only four to six PEG-5K chains by the thiolationmediated maleimide chemistry based PEGylation, another modification ofthe procedure has also been introduced. A step of capping theun-derivatized thiol functions of the Hb-PEG conjugate usingN-ethylmaleimide (NEM) has been introduced. This involves treatment ofthe reaction mixture containing PEGylated product generated after sixhours of incubation with 10 mM N-ethylmaleimide. This step is expectedto cap (derivatize) the thiols that are not modified by maleimide PEG.This product is then subjected to a tangential flow filtration stepusing Minim to isolate the PEGylated Hb free of PEG.

The physical properties of this new Hb-PEG adduct (SP-PEG5K-Hb) iscompared with that of (SP-PEG5K)₆-Hb in Table 1. Based on the RP-HPLCanalysis of the PEGylated Hb, and molecular mass analysis of thePEGylated chains, it has been calculated that new Hb-PEG adduct carriesclose to five PEG-chains (an average number). Since the Hb-PEG adduct isgenerated using a Hb concentration of 1 mM (64 mg/ml), this protocolreduces the amount of PEG reagent that needs to be used by half, ascompared to that needed for the production of the hexaPEGylated Hb.Thus, this modified protocol will reduce the cost of production ofHb-PEG conjugate, the oxygen carrying plasma volume expanders.

PEGylation of hemoglobin (Hb) by the thiolation mediated iodoacetamidechemistry based PEGylation protocol has been carried out at a proteinconcentration of 1 mM, in the presence of 10 mM iminothiolane, and 10 mMiodoacetamide PEG-5000 at 4° C. for 6 hours. The unPEGylated reactivethiols of the protein are capped with N-ethyl maleimide by reacting withthe reagent for another one hour. The product is then purified on apreparative Superose-12 column chromatography. The properties of thisnew PEG-Hb conjugate, carboxamidomethyl PEG-Hb [CAM-PEG5K-Hb], have beencompared with that of [SP-PEG5K-Hb] prepared under the same conditions,in terms of molecular radius, hydrodynamic volume, colloidal osmoticpressure (4 g %) and RP-HPLC. (Table 1). As can be seen, retention timesof these two products, the Hb-PEG-5K adducts generated by usingmaleimide-phenyl-PEG-5K and iodoacetamide PEG-5K are comparable, andslightly lower than that of (SP-PEG5K)₆-Hb (Acharya et al., 2003). Themolecular radius of the two new Hb-PEG conjugates are closer to that ofpreviously described Tetra PEGylated (PEG-5K) dog Hb than to that of(SP-PEG5K)₆-Hb. The colloidal osmotic pressure of 4 g % solution of thetwo products are around 35 mm Hg, as compared to the value of 74 mm Hgfor a 4 g % solution of (SP-PEG5K)₆-Hb.

The iodoacetamide PEG5K used in the present study introduces only analkyl (methyl) chain on the sulfur atom of the new thiol groups of thethiolated protein and the PEG. Iodoacetaimde chemistry based PEGreagents can also be used to introduce carboxamide-PEG chains on theα-amino groups and the thiol groups of Cys-93(β) of HbA without changingthe net charge on the functional groups PEGylated (conservativePEGylation), i.e. by carrying out the PEGylation reaction withcarboxamide-PEG 5K in the absence of iminothiolane.

TABLE 1 Molecular Properties of Conservatively PEGylated Hb CAM- SP-(SP-PEG-5K)₆- Hb PEG5K-Hb PEG5K-Hb Hb Molecular Radius 3.0 5.4 5.2 6.5(nm) Retention time 62 52 52 50 (min) (Superose 12) Colloidal osmotic 834 37 74 pressure (mm Hg)

III. Effect of IntraMolecular Crosslinks on Molecular Properties ofPEG-Hb Conjugate Materials and Methods

Reductive Alkylation of HbA with PEGSK-Aldehyde.

Human adult hemoglobin (HbA) was purified from human erythrocytes aspreviously described (Manjula and Acharya, 2003). αα-crosslinking HbA(αα-HbA) was prepared as previously described (Chatterjee et al., 1986).Cys-93-ββ-succinimidophenyl polyethylene glycol 2000 hemoglobin A(ββ-crosslinking HbA, PP-HbA) was prepared as described by Manjula etal. (2000). HbA, αα-HbA and ββ-HbA (0.25 mM tetramer) in 50 mMBisTris-Ac buffer (pH 6.5) were reacted with 10 mM methoxy polyethyleneglycol 5000 propionaldehyde (PEG5K-aldehyde, Shearwater Polymers,Huntsville, Ala.) in the presence of 50 mM sodium cyanoborohydride(Sigma Chemical Co., St. Louis, Mo.) at 4° C. overnight. For analyticalreactions, the reaction mixture was dialyzed extensively against PBS, pH7.4 and the product examined by size exclusion chromatographic (SEC) andRPHPLC analysis. For the preparative reactions, the reaction mixture wassubjected to diafiltration through a 70-kDa membrane vs. PBS (pH 7.4)using a Minim Tangential Flow Filtration instrument (Pall Corporation,Ann Arbor, Mich.) to remove unreacted PEG and other excess reagents. Thefinal product in the retentate was concentrated and stored frozen at−80° C.

Dynamic Light Scattering.

Dynamic light scattering for molecular radius measurement was performedusing a DynaPro instrument (Protein Solutions, Lakewood, N.J.). Samplesat the protein concentration of 1 mg/ml were centrifuged at 13,000 rpmfor 4 min prior to analysis.

Colloidal Osmotic Pressure Measurements.

A Wescor 4420 Colloidal Osmometer was used to measure the colloidalosmotic pressure of the HbA samples. The measurements were performedusing a series of concentrations of HbA samples in PBS (pH 7.4) and atroom temperature. Osmocoll reference standards were used to calibratethe instrument before measurements of the samples.

Viscosity Measurements.

The viscosity of the Hb samples was measured with a cone and a rheometerat a protein concentration of 40 mg/ml. A series of concentrations ofHbA samples were measured using the cone spindle (CPE-40, Brookfield) ata shear rate of 75 per second in PBS (pH 7.4) and at 37° C.

Analytical Methods.

Determination of the PEGylation induced size enhancement of Hb by sizeexclusion chromatography (SEC) on Superose 12 columns, RPHPLC analysisof globin chains on a Vydac C4 column (4.6×250 mm), and SDS-PAGEanalysis were carried out as previously described (Manjula et al., 2003;Rao et al., 2003). Isoelectric focusing electrophoresis (IEF) wasoperated using precast resolve gels from Isolab and a blend of pH 6-8resolve ampholytes. Gels were electro-focused for 3 h to resolve thecomponents in the sample completely. Oxygen-binding equilibriummeasurements were carried out using a Hemox Analyzer as described byCheng et al. (2002).

Tryptic Peptide Mapping.

Tryptic peptide mapping of the PEGylated Hb was carried out by methodspreviously described (Doyle et al., 1999; Lippincott et al., 1997). Thetryptic peptides were analyzed by RPHPLC on a Vydac C18 column (10mm×250 mm) using a linear gradient of 5-50% acetonitrile containing 0.1%TFA in 160 min, followed by a linear gradient of 50-70%acetonitrile-0.1% TFA in 20 min. The flow rate was 2 ml/min and theeffluent was monitored at 210 nm. Percent modification of the amino acidresidues modified in the PEGylated Hb was evaluated essentially asdescribed by Lippincott et al. (1997) and Doyle et al. (1999). Briefly,the recovery of peptide βT4 was used as an internal standard, and theratio of the peak area of each peptide of the PEGylated Hb and PEGylatedαα-Hb relative to the corresponding peak in the HbA and αα-Hb peptidemap, respectively, was used to elucidate the amino acid residuesmodified by PEGylation.

Analytical Ultracentrifugation.

Sedimentation velocity measurements were conducted in a Beckman XL-Ianalytical ultracentrifuge in PBS buffer at pH 7.4, 25° C. and 55,000rpm. Boundary movement was followed at 405 nm using the centrifuge'sabsorption optics. For each sample, data were collected at threeconcentrations (A₄₀₅=0.1, 0.5 and 1.0). The g(s*) distributions weredetermined using DCDT+ version 2.0.4 (http://www.jphilo.mailway.com)using values of v of 0.74 mUg for HbA (Kellet, 1971) and 0.806 mL/g forthe PEGylated proteins (Dhalluin et al., 2005) and normalized tostandard conditions (S_(20,W) and D_(20,W)) by correcting for bufferdensity and viscosity.

Circular Dichroism Spectroscopy.

Circular dichroism spectra of Hb samples were recorded on a JASCO-720spectropolarimeter (JASCO, Tokyo, Japan) at 25° C. with a 0.2-cm lightpath cuvette (310 μl). For the spectra from 250 to 200 nm, the Hbconcentration was 1.3 μM as tetramer. For the spectra from 480 to 250nm, the Hb concentration was 26.0 μM as tetramer. All the Hb sampleswere in PBS, pH 7.4. The molar ellipticity, θ, is expressed indeg·cm²/dmol on a heme basis.

Fluorescence Measurements.

Intrinsic fluorescence measurements of Hb samples were performed usingShimadzu RF-5301 spectrofluorimeter at room temperature. The emissionspectra were recorded from 295 to 400 nm using an excitation wavelengthof 280 nm. Excitation and emission slit widths were both 5 nm. All thesamples used were at Hb concentration of 1 mg/ml in PBS, pH 7.4. Cuvettewith 1 cm path-length was used.

Results

Influence of α-Fimaryl Intramolecular Crosslink on the Site Selectivityand Extent of PEGylation of Hb.

The sites of PEGylation as well as the extent of PEGylation of αα-Hb hasbeen established by comparing the tryptic peptide maps of unmodifiedαα-Hb with that of its PEGylated product. These results have beencompared with that of PEGylation of unmodified Hb. As presented in Table2, Val-1(α) and Val-1(β) have been completely modified by PEGylation inαα-fumaryl-Hb just as in the control uncross-linked Hb. Besides thesetwo major sites of PEGylation in the PEGylated αα-Hb, four lysineresidues also showed modification by PEGylation comparable to those ofuncross-linked proteins and these sites of partial modification(PEGylation) are also same as those in the uncrosslinked sample (Hu etal., 2005). Thus it is clear that the presence of intramolecularαα-fumary crosslink within the central cavity of Hb has essentially verylittle influence on the site selectivity of the reductive alkylationchemistry based PEGylation of the protein. Besides, the αα-crosslinkedHb has also been PEGylated to the same level of un-crosslinked Hb.Accordingly PEGylated αα-Hb is referred to (Propyl-PEG5K)₆-αα-Hb, inconformity with the earlier nomenclature of hexaPEGylated Hb generatedby the same PEGylation chemistry as (Propyl-PEG5K)₆-Hb.

Electrophoretic Analysis of (Propyl-PEG5K)₆ αα-Hb.

As shown by SDS-PAGE analysis (FIG. 2A), the electrophoretic pattern forHbA is a doublet corresponding to its α and β chain (Lane 2). The αchain of αα-Hb showed a slower mobility as a consequence ofcross-linkage (Lane 3). On PEGylation, the doublet corresponding to theunmodified globin chains disappears, and displays two major proteinbands with slower mobility relative to the unmodified globin chains andtwo minor bands that exhibit even slower mobility relative to the twomajor bands (Lane 5). For (Propyl-PEG5K)₆-αα-Hb (Lane 4), the two majorbands of (Propyl-PEG5K)₆-Hb became lighter with the concomitantappearance of two new bands with slower mobility. Iodine stain of theelectrophoresis gel to locate the PEGylated products showed that stainintensity is comparable with different bands between the two PEGylatedproteins. This suggests that the labeling of the two chains of Hb withPEG-chains is comparable. This is consistent with the results of thetryptic peptide mapping of the two PEGylated proteins.

The influence of the intramolecular crosslinking on the isoelectricfocusing (IEF) pattern of the PEGylated proteins is shown in FIG. 2B.The PEGylated proteins do not focus as compact bands, and are thusdistinct from HbA and αα-Hb. (Propyl-PEG5K)₆-Hb focused slightly behindHbA. Besides, (Propyl-PEG5K)₆-αα-Hb focused slightly behind(Propyl-PEG5K)₆-Hb. Since the reductive alkylation chemistry basedPEGylation of Hb is not expected to influence the net positive charge ofthe surface amino groups to which the PEG-chains are conjugated, it issuggested that the influence of PEGylation on the IEF pattern reflectsthe molecular shielding influence of the PEG-shell on the surfacecharges of Hb from the bulk solvent (Doyle et al., 1999). Since HbA andαα-fumaryl Hb exhibit similar isoelectric patterns, the molecularshielding influence of the PEG-shell on the surface charges of tetramermay be enhanced with (Propyl-PEG5K)₆-αα-Hb relative to that in(Propyl-PEG5K)₆-Hb.

Higher Hydrodynamic Volune of (Propyl-PEG5K)₆-αα-Hb as Compared to thatof (Propyl-PEG5K)₆-Hb by Size Exclusion Chromatography.

The molecular volume of the two PEGylated forms of Hb (crosslinked anduncrosslinked) have been compared by size exclusion chromatography (SEC)on Superose 12 column. In the lower panel of FIG. 3, the SEC patterns ofHbA and its hexaPEGylated form generated by reductive alkylation form ispresented. FIG. 3, upper panel, compares the SEC patterns of αα-fumarylHb and its hexaPEGylated form. The hexaPEGylation of HbA results in anearlier elution of the protein reflecting a significant increase in thehydrodynamic volume of Hb. The SEC pattern of HbA is not influenced bythe presence of αα-fumaryl intra molecular crosslinks. On hexaPEGylationof αα-fumaryl-Hb under the same reaction condition as that used for HbA,hydrodynamic volume of cross-linked Hb is significantly increased, muchmore than that of PEGylation of uncrosslinked HbA as reflected by theearlier elution of (Propyl-PEG5K)₆ αα-Hb as compared to(Propyl-PEG5K)₆-Hb. Based on the results of tryptic mapping (Table 2),it is evident that the larger increase in the hydrodynamic volume ofαα-fumaryl Hb compared to uncrosslinked HbA is not related to the higherlevel of PEGylation or an altered site selectivity of PEGylation. Thusαα-fumaryl intra-molecular cross-link in Hb appears to increase thepropensity of PEGylation to induce higher hydrodynamic volume. It hasbeen previously noted that the hexaPEGylated Hb exhibits a hydrodynamicvolume comparable to that of an oligomeric form of intramolecularlycrosslinked Hb that carries four tetrameric units. Thus the molecularvolume of (Propyl-PEG5K)₆-αα-Hb is higher than that of such oligomericHbs that carry four copies of intramolecular crosslinked Hbs.

Molecular Volume of (Propyl-PEG-5K)₆-αα-fumaryl Hb as Determined byDynamic Light Scattering.

The molecular radius of the various products, as determined by dynamiclight scattering and their calculated molecular volume are summarized inTable 3. The molecular radius of the αα-fumaryl Hb is comparable to thatof HbA, a result that parallels the SEC studies (FIG. 3). As reportedearlier, (Propyl-PEG5K)₆-Hb showed a molecular radius of 5.40 nm,reflecting the enhanced molecular dimensions of the PEGylated Hb.Interestingly, the molecular radius of (Propyl-PEG5K)₆-αα-Hb exhibitsfurther increase in the molecular radius as compared to(Propyl-PEG5K)₆-Hb, which lacks the αα-fumaryl crossbridge. Thecalculated molecular volume of (Propyl-PEG5K)₆ αα-Hb is nearly twicethat of (Propyl-PEG5K)₆-Hb. These results are consistent with the datafrom SEC, and quantifies the data on increase in molecular volume.

Influence of PEG-chain Length on the Molecular Volume of (Propyl-PEG)₆αα-Hb.

PEG2K-aldehyde and PEG20K-aldehyde are lower and higher homologues ofthe PEG5K-aldehyde, respectively. To establish the effect of PEG-chainlength on the influence of αα-fumaryl crosslink on the enhancedefficiency of PEGylation induced increase in the molecular volume Hb,the reductive alkylation of Hb with PEG-aldehyde has been studied usingPEG-2K aldehyde and PEG-20K aldehyde.

The hydrodynamic volume of the PEGylated products generated fromuncrosslinked and crosslinked Hb has been compared using SEC (FIG. 4A).The reductively alkylation mediated PEGylation of αα-fumaryl Hbgenerated using PEG2K-aldehyde as well as that generated using PEG-20Kaldehyde exhibited higher hydrodynamic volume as compared to therespective PEGylated product generated from uncrosslinked Hb.

FIG. 4B compares the increase in the molecular radius of Hb onPEGylation using reductive alkylation chemistry as a function ofPEG-chain length. Thus this reflects the influence of αα-fumarylcrosslinks on increase in the molecular volume resulting fromPEGylation. As the chain length of the PEG is increased, the differencein the molecular radius between the PEGylated products of crosslinkedand uncrosslinked protein is also increased. The hydrodynamic volumes ofthe three PEGylated Hbs generated from αα-fumaryl Hb is nearly twicethat of the corresponding PEGylated Hb. Therefore, the influence ofαα-fumaryl intramolecular crosslink to increase the propensity ofPEGylation to enhance molecular volume of Hb is correlated with thechain length of the PEG-aldehyde used for reductive alkylation; itreflects the size of the PEG-shell.

Influence of αα-Fumaryl Crossbridge on the Viscosity and ColloidalOsmotic Pressure of HexaPEGylated Hb.

PEGylation induced (i) enhanced molecular volume (ii) viscosity and(iii) colloidal osmotic pressure are the three important parameters thatare critical for the neutralization of vasoactivity. In view of theinfluence of αα-fumaryl intra molecular crosslinking on the molecularradius of the PEGylated Hb, its influence on viscosity and COP has alsobeen investigated and compared with that of hexaPEGylated derivativegenerated using αα-fumaryl Hb. FIG. 5 shows the correlation of COP of(Propyl-PEG5K)₆ αα-Hb with protein concentration. As with the otherPEGylated samples studied earlier, the COP of (Propyl-PEG5K)₆-αα Hbexhibits a nonlinear dependence on the protein concentration (FIG. 6).The COP of a sample reflects the number of particles in solution.(Propyl-PEG5K)₆-αα-Hb exhibited lower COP value as compared to the(Propyl-PEG5K)₆-Hb in spite of its larger molecular volume. The lowervalue of COP for the solutions of (Propyl-PEG5K)₆-αα-Hb is for theentire range of the protein concentration.

The viscosity of (Propyl-PEG5K)₆-αα-Hb at a protein concentration of 40mg/ml has been compared with that of HbA and (Propyl-PEG5K)₆-Hb and theresults are presented as an inset in FIG. 5. The PEGylation inducedincrease in the viscosity of Hb is not influenced much by the αα-fumarylintra molecular crossbridge.

Thus the presence of αα-fumaryl crossbridge (i) nearly doubles thePEGylation induced hydrodynamic volume and (ii) lowers the PEGylationinduced colloidal osmotic pressure by about 30%, but has very littleinfluence on the PEGylation induced viscosity when compared with thePEGylation induced changes in the molecular properties of uncrosslinkedHb. These influences seen as a consequence of intramolecular crosslinkscould be explained on the basis of an increase in the dissociation ofuncrosslinked Hb as a result of PEGylation as compared to that with theuncrosslinked Hb.

Analytical Ultracentrifugation Studies.

PEGylated samples as well as the starting crosslinked and uncrosslinkedmaterials have been subjected to analytical ultracentrifugation studiesto gain more insight into the influence of PEGylation on the interdimeric interactions of Hb. The samples have been subjected tosedimentation velocity analysis (FIG. 6). The decrease in sedimentationcoefficient (S) values observed for (Propyl-PEG5K)₆-αα-Hb withincreasing protein concentration is characteristic of a stablemonodisperse particle (•). The estimated M_(w) of this particle fromS^(o) _(20,w)/D^(o) _(20,w) is ˜90 kDa, consistent with a hexaPEGylatedtetramer. In contrast, the sedimentation rate of (Propyl-PEG5K)₆-Hb ismuch slower, displaying a slight decrease in sedimentation withincreasing protein concentration that lacks self-association (◯). Theestimated M_(w) of this particle is ˜60 kDa consistent with apredominant presence of PEGylated dimers. As a control, native andcrosslinked HbA was analyzed under identical experimental conditions.Crosslinked HbA also sediments as a stable monodisperse particle (•);the estimated M_(w) of ˜55 kDa is consistent with a tetramer. Incontrast, native HbA shows its well-documented dimer-tetramerassociation (◯). Comparison of the non-crosslinked Hb moleculesindicates that PEGylation destabilizes the Hb tetramer. In addition,S_(20,w) values of (Propyl-PEG5K)₆-Hb and (Propyl-PEG5K)₆-αα-Hb arelower than those of HbA and ca-Hb, indicating that the PEG-moiety can beenvisaged as a ‘parachute’ slowing down the sedimentation rate (Dhalluinet al., 2005).

Influence of αα-Fumaryl-Intra Molecular Crossbridge on StructuralFeatures of (Propyl-PEG5K)₆-Hb: (i) CD Measurements.

Possible differences between the structural features of(Propyl-PEG5K)₆-Hb and (Propyl-PEG5K)₆-αα-Hb have been investigatedusing circular dichroism spectroscopy. The far-UV (absorbance 200-250nm) CD spectra for the PEGylated proteins were shown in FIG. 7A. Asindicated by the ellipticity values at 222 nm, the α-helical content ofHbA was not changed upon the introduction of αα-cross-bridge and/orPEGylation. Thus, the secondary structure of the chains of HbA has notbeen influenced significantly either by αα-cross-linking or onsubsequent PEGylation.

In the near-UV CD region (FIG. 7B), the L-band (centered around 260 nm)is considered to be sensitive to the interactions between the heme andthe surrounding globin, being influenced by the attached ligand (Zentzet al., 1994). PEGylation of HbA induced the increase in the intensityof L band, while PEGylation of αα-Hb showed no change in the ellipticityof this band. This indicated that the increased intensity of L band ofHbA upon PEGylation was not related to PEGylation itself; but related toPEGylation induced structural changes of HbA, tetramer dissociation. Theregion around 285 nm is considered as indicative of the R to Ttransition, and correlated to the environment of α42 and β37 aromaticresidues in human HbA (Perutz et al., 1974). PEGylation of HbA and αα-Hbboth induced a decrease in the ellipticity around 285 nm associated withhigher oxygen affinity (R state) (Perutz et al., 1974). This is possiblydue to the PEGylation induced conformational change around α42 and β37.This region reflects the α1β2 subunit interface contact domain. Thus,the PEG shell of the PEGylated Hb appears to reduce the propensity ofthe molecule to transition from R to T state, and this is consistentwith the fact that PEGylation increases the oxygen affinity of Hb.

Most significant changes were noticed in the soret band region of the CDspectra. The Soret region the spectra of Hb is informative on theinteractions of heme prosthetic group with the surrounding aromaticresidues and to modifications in the spatial orientation of these aminoacids with respect to heme, affecting porphyrin transitions and π-π*transitions in the surrounding aromatic residues (Hsu and Woddy, 1971).The presence of the αα-fumaryl intramolecular crossbridge reduces theintensity of the Soret band of HbA with a wavelength shift to the red.This represents the presence of deoxy like features in the crosslinkedHb (Perutz et al., 1974). The PEGylation of Hb to generate(Propyl-PEG5K)₆-Hb increases the intensity of the soret band withoutnoticeable changes in the wavelength. This reflects that themicroenvironment of heme is slightly perturbed upon PEGylation (Hu etal., 2005). The presence of αα-crosslinks in the hexaPEGylated samplesincreases slightly the intensity of the band just as in the case ofuncrosslinked Hb, but the intensity is significantly lower than that ofthe hexaPEGylated sample without the intramolecular crosslinks. The redshift in the soret band induced as a result of the ααcrosslinking isconserved even on PEGylation, which is considered as the reflection ofthe lower affinity of heme for oxygen (Perutz et al., 1974).

Influence of αα-Fumaryl-Intra Molecular Crossbridge on StructuralFeatures of (Propyl-PEG5K)₆-Hb: (ii) Fluorescence Measurements.

Intrinsic fluorescence of Hb is primarily due to the fluorescence ofβ37Trp at the α1β2 interface, which reflects the stability of thequaternary structure of Hb (Hirsch, 2003). As can be seen in FIG. 8,when excited at 280 nm, HbA and αα-Hb showed similar fluorescenceemission intensity with a peak position at 320 nm. (Propyl-PEG5K)₆-αα-Hbalso showed comparable emission intensity to HbA and αα-Hb, indicatingthat the attachment of PEG chain did not alter the quaternaryinteractions of Hb. However, fluorescence intensity of(Propyl-PEG5K)₆-Hb is significantly higher as compared to(Propyl-PEG5K)₆-αα-Hb and exhibits a noticeable blue shift, and reflectsthe perturbation of the quaternary structure. In conjunction with theultracentrifugal studies, this may be reflective of the enhanceddissociation of the tetramers (reflection of the presence of dimers),and inhibition of such dissociation by intramolecular αα-fumaryl crossbridges.

Influence of Engineering ββ′-Succunimidophenyl PEG-2000 IntramolecularCross-Bridge on the Molecular Properties of (Propyl-PEG5K)₆-Hb.

The αα-crosslinkage engineered into (Propyl-PEG5K)₆-Hb is a centralcavity intra molecular crosslink. The central cavity of Hb plays adominant role in dictating the structural stability and functionalproperties of Hb. The influence of the αα-fumaryl crossbridge on themolecular properties of (Propyl-PEG-5K)₆-Hb may be unique as it is anαα-crosslink or it is a within the central cavity cross-bridge. In anattempt to establish the fact that the observed influence on themolecular properties of (Propyl-PEG-5K)₆-Hb is a consequence of an intramolecular crossbridge that prevents the dissociation of the tetramersinto dimers, the effect of a crosslink outside the central cavity of Hbwas investigated. Bβ′-succinimidophenyl PEG-2000 crosslink has been alsoengineered into (Propyl-PEG5K)₆-Hb to provide an answer to thisquestion. As can be seen in Table 4, the presence ofββ-succinimidophenyl PEG-2000 crosslink in (Propyl-PEG5K)₆-Hb nearlyparallels the influence of introducing the intramolecular αα-fumarylinto (Propyl-PEG5K)₆-Hb. The molecular radius and the hydrodynamicvolume are increased, there is limited influence on viscosity, and theCOP of (Propyl-PEG5K)₆-Hb decreased upon ββ-crosslinkage. Therefore,ββ-crosslinking of (PropylPEG5K)₆-Hb also achieves the same results asthe αα-crosslinking, apparently by preventing the PEGylated moleculefrom dissociating into dimers as a consequence of weakened interactionbetween the interdimer interactions.

TABLE 2 Sites of PEGylation in αα-fumaryl HbA Percent modificationResidue modified (Propyl-PEG5K)₆-Hb (Propyl-PEG5K)₆-αα-Hb Val-1(α) 100100 Val-1(β) 100 100 Lys-8(β) 23 23 Lys-11(α) 23 27 Lys-40(α) 12 11Lys-56(α) 17 22 The sites of PEGylation in the PEGylated proteins aredetermined by tryptic peptide mapping of their globin chains asdescribed previously.

TABLE 3 Molecular Radius of (Propyl-PEG5K)₆ αα-Hb Sample Radius (nm)Volume (nm³) HbA 3.14 129.6 αα-Hb 3.16 132.1 ββ-Hb 3.35 157.4(Propyl-PEG5K)₆-Hb 5.40 659.2 (Propyl-PEG5K)₆-ααHb 6.56 1181.9(Propyl-PEG5K)₆-ββ-Hb 6.70 1259.2 The protein samples at a concentrationof 1 mg/ml were centrifuged at 13,000 rpm for 4 min prior to analysis.

TABLE 4 Comparison of the Solution properties of Hexa PEGylated HbsMolecular Radius Volume COP Viscosity (nm) (nm³) (mmHg) (cp)(Propyl-PEG5K)₆-Hb 5.40 659.2 128.5 3.11 (Propyl-PEG5K)₆-αα-Hb 6.561181.9 100.8 3.23 (Propyl-PEG5K)₆-ββ-Hb 6.70 1259.2 85.2 2.97 Theviscosity and COP were measured at a Hb concentration of 4 g %. Samplesat the protein concentration of 1 mg/ml were centrifuged at 13,000 rpmfor 4 min prior to analysis.

Extension Arm Facilitated, Maleimide Chemistry Based PEGylation ofββ-Succinimodo Phenyl PEG-2000 Hb.

ββ-succinimido phenyl PEG-2000 Hb (an intramolecularly crosslinked Hb)was PEGylated using the thiolation mediated PEGylation. Its propertieswere compared with those of a nonhypertensive hexaPEGylated Hb generatedusing extension arm facilitated PEGylation of uncrosslinked Hib usingmaleimide-PEG (Table 5). In PEGylated ββ-succinimido phenyl PEG-2000 Hb,Cys-93(β) is involved in intramolecular crosslinking, and hence notavailable for PEGylation. Apparently the site selectivity of PEGylationin the crosslinked and uncrosslinked is slightly different (and theextent of PEGylation could be slightly lower). The molecular propertiespresented here show that at 4 gm % level, the extent of dissociation ofPEGylated Hb is not as significant as that seen in the PEGylated productgenerated by reductive alkylation protocol. Accordingly, to establishthis aspect further, αα-fumaryl Hb has been subjected to thiolationmediated PEGylation according to the procedures described earlier. Inthis crosslinked Hb, Cys-93(β) is accessible for reaction ofPEG-maleimide and hence the site selectivity of the product is expectedto be comparable. As seen in the Table 5, the molecular radius of theproduct generated from crosslinked Hb is slightly higher. A comparisonof the size exclusion chromatography of the PEGylated crosslinkedproduct and PEGylated uncrosslinked product as a function of the proteinconcentration (1 mM and 5 μM) has demonstrated that the productgenerated by the thiolation mediated maleimide chemistry basedPEGylation using the crosslinked Hb is larger than the product generatedfrom the uncrosslinked Hb. But the difference is smaller than that seenwith the reductive alkylation product (FIG. 9). Thus one can concludethat PEGylation of uncrosslinked Hb increased the dissociation of Hb.

With the contemplated use of hexaPEGylated product at 4 gm %, there willbe considerable in vive dilution of this sample, and the amount ofdissociated species in circulation is likely to be higher depending onthe final concentration of Hb in the plasma as compared to the amount inthe transfused solution. A further dilution of Hb concentration in theplasma as a consequence of inflow of liquid to the vascular system (aconsequence of the COP of PEGylated Hb) is also anticipated. Therefore,the use of intramolecularly crosslinked Hb is a better choice forgenerating PEGylated Hb as blood substitutes as compared to theuncrosslinked Hb that has been used earlier.

TABLE 5 Molecular properties of (SP-PEG5K)₆-Hbs Radius Volume COPViscosity Sample (nm) (nm³) (mmHg) (cp) (SP-PEG5K)6-ββ- 6.12 959.7 62.12.28 succinimidophenyl PEG-2000-Hb (SP-PEG5K)6-αα- 6.44 1118.2 — —fumaryl-Hb (SP-PEG5K)6-Hb 6.06 931.7 73.9 2.50* ββ-succinimidophenyl-3.35 157.4 PEG-2000 Hb αα-fumaryl-Hb 3.24 142.4 HbA 3.10 124.7 *Data isfrom Manjula et al. (2005)

IV. Conservative PEGylation of Albumin

PEGylated Albumin as a Plasma Volume Expander:

The simplicity of thiolation mediated maleimide chemistry basedconservative PEGylation has prompted the translation of the thiolationmediated PEGylation procedure to produce PEGylated albumin in largeamounts to facilitate its evaluation as a plasma volume expander. Giventhe fact that the molecular weight of albumin is comparable to that ofHb, hexaPEGylation of albumin by the thiolation mediated PEGylationusing maleimide PEG-5K as the PEG-reagent is likely to generate aspecies of PEGylated albumin the solutions of which will beiso-hydrodynamic volume, iso-viscosity and iso-colloidal osmoticpressure with hexaPEGylated Hb (Acharya et al., 2003).

Accordingly, thiolation mediated maleimide chemistry based PEGylation ofalbumin was carried out with maleimide-phenyl-PEG-5000 (Mal-Phe-PEG-5K),using reaction conditions comparable to that used for the PEGylation ofHb to generate non-hypertensive Hb. Albumin has 35 half-cystineresidues; all of which except one at position 34 (Cys-34) are involvedin intra-molecular disulfide bonds. Therefore, more thiol groups wereintroduced onto the protein using 2-iminothiolane to facilitate theattachment of Mal-Phe-PEG-5K chains.

Albumin has been subjected to PEGylation under two different reactionconditions to generate two PEGylated products with different levels ofPEGylation. For the preparation of both of these samples, the maleimidechemistry based PEGylation was used in the one step mode followed bytangential flow filtration of the PEGylated albumin to remove the excessPEG reagent. For the first preparation, reaction conditions wereoptimized to generate a product with an average of six PEG-chains permolecule as determined by the mass spectral analysis. The hydrodynamicvolume of the hexaPEGylated albumin is slightly larger than that of hexaPEGylated Hb. This hexa-PEGylated albumin, (SP-PEG5K)₆-albumin, exhibitsa viscosity of 2.4 cp and a colloidal osmotic pressure of 37 mm Hb.Thus, the viscosity of a 4 g % solution of hexa-PEGylated albumin isslightly lower than that of a 4 g % solution of hexa-PEGylated Hb[(SP-PEG5K)₆-Hb]. But the colloidal osmotic pressure of a 4 g % solutionof (SP-PEG5K)₆-albumin is noticeably lower than that of thehexa-PEGylated Hb.

The molecular mass of Hb and of albumin are nearly identical, but thehydrodynamic volume of albumin is slightly higher than that of Hb asreflected by size exclusion chromatography. The difference in thehydrodynamic volume is consistent with the fact that the molecularradius of albumin as determined by light scattering is around 4.0 nmwhereas that of Hb is only around 3.0 nm. Thus, the six copies of PEG-5Kchains conjugated to Hb in (SP-PEG5K)₆-Hb by thiolation mediatedconservative PEGylation are more efficient in increasing the colloidalosmotic pressure of Hb than the six copies of PEG-5K chains conjugatedby the same conjugation chemistry to albumin. Given the difference inthe molecular surface area of Hb and albumin, which is a directconsequence of the difference in the molecular radius of the twounPEGylated parent proteins, the results suggest that there is acorrelation between the number of PEG-chains of a given molecular masson a given molecular surface area, i.e., there is a correlation betweenthe density of PEG-units in a given molecular volume and the colloidalosmotic pressure of the PEGylated protein.

Another preparation of PEGylated albumin that carries on an average 12copies of the PEG-5K chains per mole, [(SP-PEG5K)₁₂-albumin], has alsobeen prepared. On size exclusion chromatography the (SP-PEG5K)₁₂-albuminelutes only slightly ahead of (SP-PEG5K)₆-albumin. Consistent with thepresence of higher number of copies of PEG-5K on the molecular surfaceas compared to that of (SP-PEG5K)₆-albumin, the viscosity and thecolloidal osmotic pressure of (SP-PEG5K)₁₂-albumin are higher, being 3.7cp and 102 mm Hg respectively, for a 4 g % solution. These resultssuggest that the correlation between the increase in the colloidalosmotic pressure of a protein as a function of the mass of PEG on agiven molecular surface is nonlinear and increases in a exponentialfashion, while the viscosity of the sample as a function of thePEGylation is more linear.

Influence of PEGylation of Albumin on its Interaction with PotentialSmall Molecular Weight Drugs:

Besides contributing to 80% of COP of plasma, albumin also acts as atransporter for insoluble fatty acids and therapeutic drugs. The bindingof drugs to albumin is very important for their pharmaco-kinetics. Dueto this binding, the clearance of drugs is slow and allows use of lowdosage of many drugs. This in turn keeps unbound drugs that can interactwith a cognate receptor at low levels and minimizes side effects. Inorder to determine the ligand binding capability of PEGylated albumin,the binding of a therapeutic drug, warfarin has been studied. Warfarinbinds to albumin at Site-I binding site that is located in the A domainof the protein. The binding constants for albumin and(SP-PEG5K)₁₂-albumin has been found to be 3.68 and 3.1×10⁻⁵ M⁻¹,respectively. Thus, thiolation-mediated PEGylation of albumin does notimpair its drug binding capability of site-I.

The propensity of the PEGylation reaction to endow PEGylated albuminwith an enhanced molecular radius besides increased viscosity andcolloidal osmotic pressure (COP) should lower filtration. Thus, theextravasation seen on transfusion with albumin solutions, particularlyin some pathological conditions, should be reduced. Given the increasedviscosity and COP, a lower concentration of PEGylated albumin needs tobe used (relative to albumin) to maintain the same parameters.

(SP-PEG5K)₆-albumin and (SP-PEG-5K)₁₂-albumin with enhanced molecularsize (hydrodynamic volume), viscosity and colloidal osmotic pressure canserve as better plasma expanders than albumin itself, especially undersome pathological conditions wherein there is an increase in theleakiness of the blood vessels for albumin causing edema. Since the drugbinding activity of these PEGylated albumin are not significantlyinfluenced by PEGylation, these products are expected to function betterthan other conventional crystalloid or colloidal plasma expanders thatare currently in use.

V. Thiocarbmoyl PEG Albumin

Human serum albumin (0.5 mM) in 10 mM phosphate buffer was reacted with5 mM (10 fold molar excess) of isothiocyanato phenyl PEG 5K at 4° C.either at pH 6.5 or at pH 9.2 overnight. The reaction mixture wassubjected to tangential flow filtration against phosphate bufferedsaline, pH 7.4, to remove the excess PEG reagent. The removal of the PEGreagent from the sample was followed by an FPLC analysis that monitoredthe absorbance at 210 nm and the refractive index of the effluent. Thesample thus generated exhibited a high degree of molecular sizehomogeneity. The sample generated at pH 6.5 had about four copies ofPEG-5K chains while the one generated at pH 9.2 carried nearly 6 to 7copies of PEG-5K chains.

VI. Surface Decoration of Human Serum Albumin (HSA) with Multiple Copiesof Polyethylene Glycol 5000 (PEG5K) Chains: Extension Arm FacilitatedConservative PEGylation

The reactivity and accessibility of the surface functional groups ofproteins to macromolecular PEG-reagents are the two major factors thatinfluence efficiency of PEGylation of proteins. A thiol-maleimidechemistry based, extension arm facilitated PEGylation protocol has beendeveloped to overcome these limitations and applied to HSA to developPEGylated HSA as a plasma volume expander. By controlling theconcentration of HSA and of iminothiolane (a reagent that engineers theextension arm on surface amino groups with a thiol group at its distalend) the number of copies of PEG-chains coupled to HSA can becontrolled. HexaPEGylated HSA has been generated to compare thechemical, biochemical and colligative properties of the material withthat of vasoinactive, nonhypertensive hexaPEGylated Hb (Manjula et al.,2005). The one step PEGylation protocol wherein the maleimide PEG ispresent during thiolation is an improved protocol that avoids formationof thiolation induced oligomerization of HSA during PEGylation.Interestingly, PEGylation induced properties of hexaPEGylated HSA aredistinct from those of hexaPEGylated Hb. The lower viscosity andcolloidal oncotic pressure and higher hydrodynamic volume of PEGylatedHSA compared to the hexaPEGylated HbA at comparable proteinconcentrations suggests that either the colligative properties ofPEGylated protein are a correlate of the density of the PEG-chains onthe molecular surface, or hexa PEGylated Hb dissociates to have a highernumber of effective particles in solution. The simplicity and costeffectiveness of this PEGylation protocol makes this a candidate forlarge scale production of PEGylated HSA as a plasma volume expander.

VII. Thiolation of HexaPEGylated Albumin

Thiocarbamoyl human serum albumin exhibits molecular propertiescomparable to that of hexaPEGylated human serum albumin generated byextension arm facilitated PEGylation, and is a good plasma expander justas the hexaPEGylated albumin that was generated by the extension armfacilitated maleimide chemistry based PEGylation. Accordingly,additional molecular properties can be introduced to PEGylated albuminto increase its clinical applications.

One such contemplated molecular property is to facilitate the transportof nitric oxide (NO) when the PEGylated albumin is in the plasma, i.e.to facilitate the transport of NO by PEGylated albumin by generatingPEGylated and thiolated human serum albumin, where the thiol groups cantransport NO as S-nitroso derivatives. Transport of NO by the proteinthiols is an established mechanism for the transport of the oxygen.Reaction of iminothiolane with thiocarbomoyl PEG albumin generated thedesired PEGylated thiolated human serum albumin. FIG. 10 presents thekinetics of thiolation of the PEGylated protein with iminothiolane at aprotein concentration of 0.5 mM and an iminothiolane concentration of 10mM at pH 7.4 and 4° C. Stopping the reaction at a given time pointgenerates the PEGylated product with the desired level of thiol groups.By increasing the concentration of iminothiolane to 40 mM, as many asnearly 20 thiol groups could be introduced per molecule.

Another molecular property that has been engineered into the PEGylatedalbumin is an added ability for the PEGylated molecule to scavenge freeradicals in the circulatory system. Increased levels of free radicals isthe consequence of oxidative stress in biological systems.Polynitroxylation of proteins is an approach that has been advanced tofacilitate the scavenging of such free radicals. Covalent attachment ofnitroxyl radicals is engineered onto PEGylated protein by the thiolationmediated maleimide chemistry based approach; a polynitroxylatedPEGylated albumin has been prepared by reacting PEGylated albumin withmaleimide proxyl (or maleimide tempol) in the presence of iminothiolane.In the present study, PEGylated polynitroxylated albumin has been nowgenerated. Thus, these PEGylated albumin molecules can achieve bothvasodilation and scavenging of free radical when in the circulation.

VIII. Thiolation Mediated Non-Conservative PEGylation of Proteins

The ε-amino groups of the Lys residues of proteins can also be thiolatedusing bis succinimidyl dithio propionate (DTSP) or dithiosulfosuccinimidyl propionate (DTSSP) (FIG. 11). In this protocol, theprotein is thiolated at the ε-amino groups by the acylation chemistryand accordingly the thiolation of the protein is accompanied by a lossof the positive charge of the ε-amino groups derivatized. The protein isfirst derivatized with the bifunctional disulfide bridged active esterunder conditions wherein, predominantly, a monofunctional modificationof the protein is accomplished. The excess reagent is separated from themodified protein, and the modified protein is converted to a thiolatedprotein in the presence of maleimide using non-thiol reducing agents,for example, tris carboxyethyl phosphine. This thiolation mediatedPEGylation protocol has been developed to generate a new class ofPEGylated proteins.

Since the solution properties of the PEGylated protein are not a directconsequence of PEG mass, the charge of the amino group influences theconsequence of PEGylation. Accordingly, one can choose the PEGylationstrategy to manipulate (customize) the solution properties of PEG-Hb andPEG albumin.

Instead of using dithiosuccinimidyl propionate (acylation chemistry),one could use dithiobispropionimidate (amidation chemistry) to achievethe thiolation without altering the positive charge at the site of theattachment of the extension arm.

The flexibility of the thiolation mediated PEGylation protocol can beincreased by using other functionalized PEG regents specific forsulfhydryl groups, e.g. iodoalkylamide PEG derivatives, vinyl sulfonePEGs and mixed disulfides of PEG.

Besides PEGylated albumin generated by thiolation mediated PEGylation,PEG-albumin has also been generated using isothiocyanato chemistry basedPEGylation.

Both the conservative and non-conservative thiolation protocolsdiscussed in this disclosure engineer an “extension arm” between theprotein and the PEG-chain as compared to the simple nonconservativePEGylation that involves the formation of an isopeptide bond (PEGconjugating group) directly on the ε-amino group of the protein. The“extension arm” introduced between the PEG and protein appears to reducethe propensity of the PEG chain to endow the PEGylated protein with ahigher viscosity and colloidal osmotic pressure.

The engineering of the extension arm also increases the accessibility ofthe newly introduced thiol groups. The flexibility of the thiolationprotocol can be increased to manipulate the solution properties ofPEGylated protein and/or accessibility of the new thiol groups byvarying the length of the extension arm from 3 to 4, six to eight carbonatoms by using propioic acid, butyric acid, caproic acid or caprylicacid as the extension arm, using either acylation or amidation chemistryto attach the extension arm that has a thiol group at the distal endprotected either as a symmetrical disulfide of a mixed disulfide.Dithiopyridyl group is used to generate a mixed disulfide that can beused for protein thiolation.

The general structure of the reagent that can be used for theengineering of the ‘extension arm’ is a thiopyridyl succinimidyl(sulfosuccinimidyl) derivative or thiopyridyl imidate ester of omegamercapto derivative of an aliphatic carboxylic acid, and is representedby a general structure

X—(CH₂)n-S—S—Py

wherein X could be a succinimidyl carboxylate or sulfosuccinimidylcarboxylate or an imidate, n=2, proponic acid derivative, n=2, butyricacid derivative, n=5, caproic acid derivative, n=7, caprylic acid, orany other omega mercapto aliphatic acid depending the desired length ofthe extension arm. When acylation chemistry is used for linking theextension arm to the protein, as the length of the alkyl chain of theextension arm increases, it may be useful to employ sulfosuccinimdylderivative as the sulfo derivative is likely to be more soluble than thesuccinimidyl derivative. The amidation chemistry will attach theextension arm to the surface amino group without altering the net chargeof the protein (conservative modification); the acylation chemistrymediated linking of the extension arm alters the net surface charge ofthe protein (non-conservative modification).

IX. Discussion

PEGylation, the conjugation of PEG-chains to proteins, has become a veryuseful approach in biotechnology in the development of peptide andprotein therapeutics. The conjugation of a given PEG mass to apeptide/protein could be achieved by either conjugating multiple copiesof small PEG-chains or a limited number of larger PEG-chains. Thepreferred pattern of PEGylation appears to be dictated by the functionthat the PEGylated protein is expected to perform in vivo.

Thiolation mediated maleimide chemistry based PEGylation increases theaccessibility of the surface amino groups of proteins for PEGylationthrough the step of thiolation by engineering ‘extension arms’ on thereactive surface amino groups of proteins. The thiolation step alsomakes it possible to use the very selective maleimide chemistry forPEGylation of proteins. On reaction of the protein with 2-iminothiolane(8-mercapto butyrimidation), new thiol groups are introduced at thereactive ε-amino groups at a distance of four carbon atoms(approximately 9 to 10 Angstroms) away from the original nitrogen atomof the amino group. The new thiol groups are used as the target sitesfor the very selective maleimide chemistry based conjugation with PEG.

Albumin and Hb have been now subjected to PEGylation using a modifiedthiolation mediated PEGylation protocol that involves the capping stepusing N-ethyl maleimide at the end of the PEGylation reaction.Properties of the PEGylated albumin and PEGylated Hb generated by themodified thiolation mediated protein PEGylation platform have beendescribed, along with the potential therapeutic application of PEGylatedalbumins as a plasma volume expander and that of PEGylated Hbs as anoxygen carrying plasma volume expander.

The thiolation of proteins based on the amidination of the reactiveε-amino groups and subsequent PEGylation does not neutralize theoriginal positive charge of the ε-amino groups; accordingly thisapproach is referred to as conservative PEGylation. The iminothiolanemediated thiolation of proteins has been coupled with alkylationchemistry to develop a thiolation mediated alkylation chemistry basedPEGylation as an alternate conservative PEGylation protocol.

A non-conservative thiolation protocol based on acylation chemistry hasalso been developed. In this case the thiol groups are introduced on thereactive surface amino groups by acylation using a disulfide basedbifunctional reagent and subsequent reduction of the disulfide bond toexpose free thiol groups on the proteins. These thiols can then bePEGylated using either PEG maleimide or iodoactamide PEG. The acylationchemistry based thiolation of the amino groups is accompanied by theloss of the positive charge of the amino group. Therefore, acylationchemistry based thiolation of protein followed by PEGylation is referredto as nonconservative thiolation mediated PEGylation. The same principlecould be used using bis dithiopropinimidate, wherein the thiolation canbe achieved by the amidation chemistry just as the iminothiolationmediated PEGylation, which will be again conservative thiolationmediated PEGylation. The thiolation mediated (i) succinimidylationchemistry based and (ii) alkylation chemistry based PEGylationstrategies in the conservative as well as nonconservative modes areexpected to facilitate the design of new plasma volume expanders andoxygen carrying plasma volume expanders.

The general principles of the thiolation based PEGylation will alsofacilitate the conjugation of multiple copies of other sulfhydryl groupspecific reagents as well as PEG reagents, mixed disulfides of thiolPEG, and PEG vinyl sulfone to proteins. Similarly, dextran andhydroxyethyl starch plasma volume expanders could also be coupled toproteins using the thiolation based platform by derivatizing these intosulfhydryl group specific reagents. The hybrid albumin/Hb products withunique properties could be produced for special clinical applications.

PEGylation of Hb has turned out to be an alternate approach to overcomethe vasoactivity of acellular Hb, and this is achieved by making thesolution of Hb a plasma volume expander. The efficiency of albumin,which itself has been used as a plasma volume expander, is significantlyenhanced on PEGylation, and PEGylation of albumin also induces some newclinical properties to Hb. During the course of comparison of themolecular properties of PEGylated albumin and PEGylated Hb, it was seenthat efficiency of increasing colloidal osmotic pressure is better withHb than with albumin, even though both proteins have similar molecularmass. This prompted an investigation of the influence of the presence ofan intramolecular crossbridge into Hb on the PEGylation inducedmolecular properties and structural features of Hb.

The three PEGylation induced molecular properties of Hb that haveconsidered as the players in the neutralization of the vasoactivity ofHb are (i) enhanced molecular volume (hydrodynamic volume), (ii)viscosity and (iii) colloidal osmotic pressure. The increase in themolecular volume of Hb resulting from PEGylation has been quantitated bythree different approaches: (i) by calculation of the molecular radiusbased on the colloidal osmotic pressure, (ii) by size exclusionchromatography of the PEGylated Hb on superose-12 columns and (iii) bydetermination of molecular radius by dynamic light scattering. Themolecular radius of Enzon PEGylated Hb was calculated by Vandegriff andher colleagues based on the colloidal osmotic pressure of PEGylatedbovine Hb, and a value of 15 nm was assigned to this decaPEGylatedbovine Hb. The non-hypertensive PEGylated Hb generated by extension armfacilitated PEGylation [(SP-PEG-5K)₆-Hb] (Acharya et al., 2005) wasestimated to be around 14 nm when calculated based on the colloidalosmotic pressure. However, the value of this hexaPEGylated Hb asdetermined by dynamic light scattering is around 6 nm (Manjula et al.,2005). The hexaPEGylated Hb generated by reductive alkylation chemistryis around 5.5 nm as determined by dynamic light scattering. Consistentwith this the hydrodynamic volume of two hexaPEGylated Hb generated byusing two different chemistries is nearly the same as reflected by sizeexclusion chromatography (Hu et al., 2005). This hydrodynamic volume ofhexaPEGylated product is comparable to that of the oligomer of Hb thatcarries four copies of the tetramer, which is generated by theintermolecular crosslinking of Hb.

Given the fact that the colloidal osmotic pressure of(Propyl-PEG-5K)₆-Hb is nearly the double that of (SP-PEG-5K)₆-Hb (Hu etal., 2005), the molecular radius of this hexaPEGylated Hb if calculatedbased on its COP will be even higher than that of decaPEGylated bovineHb of Enzon. This is thought to reflect the influence of chemistry ofPEGylation (conjugation) on the PEGylation induced colloidal osmoticpressure of the protein. The hexaPEGylated Hb generated by acylationchemistry that neutralizes the positive charge of the surface aminogroups of Hb was even higher than that of (Propyl-PEG5K)₆-Hb. Thus,there appears that there is no correlation between the PEGylationinduced size enhancement (molecular radius) and COP.

The present study revealed that the intramolecular crossbridge has asignificant influence on the PEGylation induced increase in themolecular volume and decrease in the colloidal osmotic pressure. Themolecular volume increase on hexaPEGylation of Hb is nearly doubled bythe presence of intramolecular crosslinking. On the other hand, the COPis lowered at least by 30 to 35% by the presence of intramolecularcrosslinks. These two influences are independent of the chemistry andthe location of intramolecular crosslinks. Both inside the centralcavity, very short and rigid crossbridges, as well as outside thecentral cavity, flexible and long crossbridges, exhibited nearlyidentical responses, suggesting that the prevention of the dissociationof the PEGylated Hb into dimers is the primary molecular aspect for themodulation of the PEGylation induced molecular properties of Hb.

Confirmation of the influence of intra molecular crossbridges inincreasing molecular size of PEGylated Hb comes from the analyticalultracentrifugation studies. An increase in the molecular size reflectsthe prevention of the dissociation of the PEGylated tetramer. Acomparison of the S-values of the PEGylated Hb and PEGylated αα-fumarylcrossbridged Hb clearly showed that the two molecules are very distinctin terms of the S-values, with the crossbridged molecule exhibiting ahigher S-value. The M_(w) value of the PEGylated crossbridged moleculeis ˜90 K, while that of the PEGylated uncrosslinked Hb is around ˜60 K,reflecting the smaller molecular mass of the PEGylated uncrosslinkedmaterial. The concentration dependence of S values, confirms thathexaPEGylation of uncrosslinked Hb increases the dissociation of thetetramer into dimers. It should also be noted that the S values of thePEGylated molecules are lower than the corresponding parent molecules,even though a mass of nearly ˜30 K has been conjugated to the tetramer.Thus PEGylation of Hb increases the resistance of the molecule tosediment, and this may be an important aspect of the molecule in termsof the biological significance of Hb. In this respect, the behavior ofthe PEGylated Hbs may be compared to that of lipoproteins that arecharacterized by their floatation patterns.

Thus, the consequence of PEGylation, particularly in terms of themolecular properties that could be endowed to the molecule is influencedby the presence of intramolecular crossbridges. The significantlyenhanced molecular volume and the lower colloidal osmotic pressure of(Propyl-PEG5K)₆-αα-Hb relative to the (Propyl-PEG5K)₆-Hb makes thecrosslinked Hb a better substrate than uncrosslinked Hb for bloodsubstitutes. The higher molecular volume of the PEGylated crosslinked Hbwill further reduce the slow, but nonetheless possible, extravasation ofPEGylated uncrosslinked Hb. In addition, the lower COP of the PEGylatedcrosslinked Hb relative to that of PEGylated uncrosslinked Hb makes itpossible to use a higher concentration of Hb, without the possibledilution of the infused Hb by an increase in flow of fluids from theinterstitial tissues to the vascular system. This has been the majorlimitation of the current versions of PEGylated Hbs in attempts toincrease the level of tissue oxygenation. The absence of significantinfluence of intramolecular crosslinks on the viscosity of PEGylated Hbsuggests that the viscosity of PEGylated Hb is a direct correlate of thePEG conjugated to protein (protein to PEG ratio). However, it should benoted that the viscosity as well as COP of a mixture of methoxy-PEG(amount being comparable to that of hexaPEGylated Hb) and Hb is very lowas compared to that of PEGylated Hb. This reflects the need for thecovalent attachment of PEG-chains to Hb to induce the molecularproperties discussed here. This may reflect the fact that onconjugation, the PEG is defined in a defined domain surrounding theprotein core (PEG-shell), and it will be hard if not impossible toachieve such a high concentration of PEG in the solution to mimic thateffect.

A very interesting result of the present study is that the rate ofsedimentation of Hb is reduced on conjugation of PEG-chains to theprotein, even though the molecular mass of the PEG-Hb conjugate ishigher than that of the unmodified sample. This is apparently thecontribution of the PEG-shell around the protein core which lowers therate of sedimentation of the conjugate. This propensity of theconjugated PEG to reduce the rate of sedimentation of the protein isindependent of the presence of intramolecular crosslinks. The majorconsequence of conjugation of multiple copies of PEG-5K chains to Hb isan unusual enhancement in the molecular volume of the protein to thegiven the mass of PEG-chain conjugated. This results in a very lowdensity of atoms in the PEG-shell relative to that in the protein core.During sedimentation analysis, reorientation of the low density PEGshell causes the covalently attached PEG-chains to behave as aparachute, increasing the hydrodynamic drag on the molecule and slowingdown the rate of sedimentation. It is conceivable that similar influenceis exerted by the PEG-chains when the PEG-Hb is used as blood substituteand introduced into the circulatory system. In this situation theinteraction of the reoriented PEG-chains with the endothelium at theblood tissue interface may provide an additional mechanical stimulusthat is different from that due to shear stress developed on theendothelial surface determined by the local shear rate and the bulkviscosity of the medium. The potential role of PEGylated proteins inproviding additional mechanism of interaction with the endothelium hasimportant physiological/biological consequences because it would allowlowering overall viscosity while maintaining the necessary level ofmechanical stimulation of the endothelium, necessary for mechanotransduction mediated homeostasis. A direct practical consequence oftheses findings is the development of these PEGylated proteins as newand effective plasma volume expanders.

A major finding of the present work is that PEGylated uncrosslinked Hbgenerated by reductive alkylation chemistry predominantly exits asdimers as reflected by size exclusion chromatography. A comparison ofthe SEC of the crosslinked and uncrosslinked PEGylated products at twoconcentrations (5 μM and 1 nM) showed that the same difference exits inthe hydrodynamic volume of two PEGylated proteins at both proteinconcentrations tested. Nonetheless it should be noted that during SEC,there is a degree of dilution of the sample, which may contribute to theshift in the equilibrium between the tetramer and the dimers as comparedto that in the bulk solution. However, the predominant presence ofdimeric forms of (Propyl-PEG5K)₆-Hb in 4 gms % solution of the productis reflected by the COP data.

The influence of the crosslinks in the PEGylated Hb is also reflected inthe circular dichroic spectra and fluorescence spectra of the products.The CD measurements reflect the perturbation of the heme environment andthe fluorescence data suggest perturbation of the α₁β₂ interface of Hbby PEGylation, and a reduced effect of PEGylation on these structuralaspects by the presence of αα-fumaryl crossbridge. Thus, even from thestructural point of view, the intramolecular crosslinked Hbs appear tobe a better substrate for PEGylation to generate PEGylated Hb as bloodsubstitutes.

The molecular basis for enhancing the dissociation of Hb tetramers intodimers upon PEGylation is also of interest from a structural point ofview. Typically, association of dimers into tetramers is drivenprimarily by formation of the α1β2 interface that involves more polarcontacts between the C and N termini and the C-helices and FG corners ofboth subunits. Since the complete modification of N-termini has takenplace in the reductive alkylation chemistry, this can influence theinteractions at both the as and the ββ-ends of the central cavity. Inaddition, the association of αβ dimers to tetramers is facilitated byelectrostatic attraction between positively charged α subunits andnegatively charged β subunits. The new hydrated PEG-shell around theprotein core generated by the covalently attached PEG chain to Hbmolecule can also shield the charge of α and β subunits, which in turncan decrease the intersubunit electrostatic attractions either throughperturbation of the hydration within the central cavity or a directconsequence of the perturbation of the hydration shell of Hb by thepresence of the new hydrated PEG-shell.

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1. A PEGylated hemoglobin or a PEGylated albumin comprising apolyethylene glycol (PEG) conjugated to hemoglobin or to albumin,wherein the PEG is a maleimide PEG, an alkylamide PEG, an iodoacetamidePEG, a p-nitro thio-phenyl PEG, a vinyl sulfone PEG, or a mixeddisulfide PEG.
 2. The PEGylated hemoglobin of claim 1, wherein thePEGylated hemoglobin is a carboxamidomethyl (CAM) PEGylated hemoglobin.3. The PEGylated hemoglobin or PEGylated albumin of claim 1, wherein themaleimide PEG is a maleimide phenyl PEG or a maleimide PEG comprising analkyl linker.
 4. The PEGylated hemoglobin or PEGylated albumin of claim1, wherein the PEG is attached to the albumin or to the hemoglobin via alinker and/or an extension arm.
 5. The PEGylated hemoglobin or PEGylatedalbumin of claim 4, wherein the linker comprises an alkyl, aryl and/orheteroaryl group. 6-8. (canceled)
 9. The PEGylated hemoglobin orPEGylated albumin of claim 1, wherein each PEG chain has a molecularweight of 3,000 to 5,000 daltons. 10-14. (canceled)
 15. The PEGylatedalbumin of claim 1, wherein 6-18 PEG chains are conjugated to albumin.16-18. (canceled)
 19. The PEGylated hemoglobin of claim 1, wherein 2-8PEG chains are conjugated to hemoglobin.
 20. (canceled)
 21. ThePEGylated hemoglobin or PEGylated albumin of claim 1, wherein PEGylationdoes not alter the surface charge of albumin or hemoglobin.
 22. ThePEGylated hemoglobin or PEGylated albumin of claim 1, wherein PEGylationdoes alter the surface charge of albumin or hemoglobin.
 23. (canceled)24. The PEGylated hemoglobin of claim 1, wherein the hemoglobin containsan intramolecular crosslink.
 25. The PEGylated crosslinked hemoglobin ofclaim 24, wherein crosslinking the hemoglobin increases molecular volumeof the PEGylated crosslinked hemoglobin.
 26. (canceled)
 27. A method ofpreparing a PEGylated hemoglobin or a PEGylated albumin comprising; a)reacting hemoglobin or albumin with a thiolating agent to producethiolated hemoglobin or thiolated albumin; b) reacting the thiolatedhemoglobin or the thiolated albumin with a PEGylating agent; and c)capping unPEGylated reactive thiols of hemoglobin or albumin withN-ethyl maleimide.
 28. (canceled)
 29. The method of claim 27, whereinthe PEGylating agent is an iodoacetamide PEG or a maleimide PEG.
 30. Amethod of preparing a PEGylated hemoglobin or a PEGylated albumincomprising; a) reacting hemoglobin or albumin with dithio sulfosuccinimidyl propionate (DTSSP) or with dithiosuccinimidyl propionate(DTSP) or with or dithiobispropionimidate to thiolate the hemoglobin oralbumin, and b) reacting the thiolated hemoglobin or the thiolatedalbumin with a PEGylating agent.
 31. (canceled)
 32. The method of claim27, wherein the hemoglobin contains an intramolecular crosslink.
 33. APEGylated hemoglobin or a PEGylated albumin prepared by the method ofclaim
 27. 34. A composition comprising the PEGylated hemoglobin or thePEGylated albumin of claim 1 and a pharmaceutically acceptable carrier.35. A blood substitute comprising the PEGylated hemoglobin or PEGylatedalbumin of claim
 1. 36. A method of treating a subject which comprisesadministering to the subject a PEGylated hemoglobin or a PEGylatedalbumin of claim 1.